RNA synthesis from RNA templates in eukaryotic systems remains an ill-defined process. During infection of mammalian cells with poliovirus, an RNA-dependent RNA polymerase activity is induced which catalyzes the replication of the viral genome and the production of messenger RNA. The initiation of viral RNA synthesis in this case is unique due to the presence of a protein, VPg, on the 5' -end of the virion RNA. This proposal is directed at two facets of this RNA synthesis. (1) The replicative structures produced in infected cells will be examined. Replicative intermediates (RI) as they exist in vivo will be analyzed by covalently-crosslinking with psoralen derivatives in vivo followed by observation of purified, denatured RI in the electron microscope. A comparison of the proteins covalently-attached to virion RNA (plus strands) and to complementary RNA (minus strands) will be made by protein and peptide analyses. Also, elucidation of the sequence at the 3' -terminus of minus strands by standard RNA sequencing procedures will provide information about the potential site of initiation of plus strand synthesis. (2) A polio-specific RNA polymerase activity which can initiate synthsis and elongate to produce genome-length polio RNA strands in vitro will be studied. These studies will include an analysis of the 5' -terminus of the product synthesized from an exogenous template, identification of infected cell activities which can donate VPg to the minus 5' -terminus of product strands, and identification of viral-specific polypeptides essental for initiation of RNA synthesis. These studies will provide significant insight into the mechanism of RNA-directed RNA synthesis.